REGULATION OF DNA REPLICATION

SOS in Biochemistry, Jiwaji University, Gwalior

M.Sc. II Semester (2019-20)

Paper BCH 201: Fundamentals of Molecular Biology (Unit 1)

Replication Occurs in 3 Stages

• Initiation: begin at a specific site,

e.g. oriC for E. coli.

• Elongation: movement of the

replication fork

• Termination: at ter sites for E. coli

Replicon = unit that controls replication

Replicator: cis-acting DNA sequence required for initiation;

defined genetically

Origin: site at which DNA replication initiates; defined

biochemically

Initiator: protein needed for initiation, acts in trans

Regulation of Replication of

Bacterial Plasmid DNA

Occurs at Initiation of Plasmid DNA Replication

Two major mechanisms of control

of initiation have been recognized:

1. Regulation by antisense RNA (e.g., ColE1-derived plasmids, restricted host range)

2. Regulation by binding of essential proteins

to repeated sequences called iterons (e.g., pSC 101-derived plasmids, broad host range)

Regulation by Antisense RNA

Regulation of Replication of Col E1-derived Plasmids

Regulation by Iteron Sequences

Origin Region of pSC 101

R1, R2 and R3 are the three iteron sequences.

IR1 & IR2 are inverted terminal repeats → RepA binds and autoregulates its own synthesis

Regulation of Replication of

Bacterial Genomic DNA

Occurs at Initiation of Replication of Bacterial Genome

REGULATION OF DNA REPLICATION

1. Replication in bacteria and in eukaryotes is licensed and

permitted to occur only once per cell cycle.

2. Each replicon is allowed to fire only once per cell cycle

3. What mechanisms are in place to ensure re-initiation

does not occur in the same cell cycle?

4. Because it is critical to maintain genomic integrity,

multiple mechanisms exist to ensure that each replicon

fires once and only once during each cell cycle.

5. Three mechanisms are worth emphasizing over here:

a) Regulatory inactivation of DnaA (RIDA)

b) DnaA titration

c) Sequestration

DnaA

Only used at initiation

It plays important role in regulation

It is sometimes called as licensing factor in bacteria.

Mutations cause a slow-stop phenotype

Binds to the 4 copies of 9 bp repeats

Further cooperative binding brings in 20 to 40 DnaA monomers

Melts the DNA at the 3- 13 bp repeats

Proteins needed for initiation at oriC

Replication

Begins

DnaA

1. ~1000 DnaA molecules are present in each E. coli are

stable.

2. DnaA∙ATP → active

3. DnaA∙ADP → inactive

4. Intracellular DnaA∙ATP levels rise and fall during the

cell cycle

5. ~80% of the DnaA is present in the active DnaA∙ATP

level just prior to the initiation but falls to about 20%

after replication is initiated.

6. There is no cyclic variation in the overall concentration

or expression of DnaA.

Q/- How that happens?

Ans: Via different mechanisms.

Three mechanisms are worth emphasizing

over here:

a) Regulatory inactivation of DnaA (RIDA)

b) DnaA titration

c) Sequestration

Methylation of Bacterial Origin

Regulates Replication during

Initiation

Functions of DNA methylation in

bacteria are dual

1. To regulate DNA replication at the initiation

level

2. To distinguish between old template strand

from newly synthesized strand during DNA

mismatch repair

Regulation of replication by

methylation

G Am

T C

C T Am

G

G Am

T C

C T A G

G A T C

C T Am

G

G Am

T C

C T A G m

G Am

T C

C T Am

G

replicate methylate (lags behind replication)

Fully methylated Hemimethylated

dam methylase

Fully methylated

Will replicate Will not replicate Will replicate

CONTROL BY METHYLATION

• GATC motifs are substrates for methylation by

dam methylase.

• Methylase transfers a methyl group from S-

adenosylmethionine to N-6 of adenine in GATC.

• Methylated GATC on BOTH strands: oriC will

serve as an origin

• Methylated GATC on ONLY one strand

(hemimethylated): oriC is not active

• Re-methylation is slow, delays use of oriC to

start another round of replication.

SUMMARY

1. The full scope of the system used to control re-

initiation of replication in the same cell cycle is

not clear.

2. But several mechanisms may be involved:

a) Physical sequestration of the origin

b) delay in remethylation of origin

c) Inhibition of DnaA binding

d) Repression of dnaA transcription

3. It is not immediately obvious which of these

events cause the others, and whether their

effects on initiation are direct or indirect.

REGULATION OF REPLICATION

OF EUKARYOTIC DNA

1. A chromosome is divided into many replicons

2. The progression into S phase is tightly controlled

3. Each replicons are ~40 (yeast/fly) to 100 kb (animal)

in length

4. The origin in each replicon is activated once and

only once in a single division cycle

5. Individual replicons are activated at characteristic

timing during S phase

6. Regional activation patterns suggest that replicons

near one another are activated at the same time

7. Available evidences suggest that most chromosomal

replicons do not have a termination region like

bacteria

8. Replication fork continues from its origin until it

meets a fork proceeding towards it from the

adjacent replicon

Each Eukaryotic Chromosome Contains Many Replicons